Apparatus for encapsulating biological active substances into erythrocytes

ABSTRACT

This invention relates to a process for the encapsulation in human or animal erythrocytes of at least one substance having a biological activity, characterized in that the primary compartment of at least one dialysis element is continuously supplied with an aqueous suspension of erythrocytes, the secondary compartment of the dialysis element contains an aqueous solution which is hypotonic with respect to the erythrocyte suspension in order to lyse the erythrocytes, the erythrocyte lysate is in contact with said substance having a biological activity and, in order to reseal the membrane of the erythrocytes, the osmotic and/or oncotic pressure of the erythrocyte lysate is increased after it has been brought into contact with said substance having a biological activity.

This is a division of application Ser. No. 546,015, filed Oct. 27, 1983,now U.S. Pat. No. 4,652,449, which is a continuation-in-part of Ser. No.470,817, Feb. 28, 1983, abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention which was realized in collaboration with the CENTRENATIONAL DE LA RECHERCHE SCIENTIFIQUE, the CENTRE REGIONAL DETRANSFUSION SANGUINE represented by the CENTRE HOSPITALIER REGIONAL DETOURS, and the STUDIENGESELLSCHAFT KOHLE mbH, relates to a process forthe encapsulation in human or animal erythrocytes of at least onesubstance having a biological activity, to an apparatus which may beused for carrying out this process and to the erythrocytes which areobtained.

BACKGROUND INFORMATION

Before describing the present invention, it may be advantageous torecall some concepts of the subject matter.

Blood is a complex mixture consisting of a liquid, the plasma, in whichare suspended red cells (erythrocytes) which transport oxygen, whitecells (leucocytes) and platelets. The plasma contains solubilizedsoluble metabolites, proteins and salts which are exchanged in thedifferent tissues.

Only erythrocytes are of interest within the scope of the presentinvention.

In a simplified manner, it may be considered that erythrocytes consistof a membrane which surrounds a cytoplasm which is essentially filledwith haemoglobin.

The function of haemoglobin is to combine with molecular oxygen duringthe passage of the erythrocytes through the lungs and to transport thisoxygen to all the tissues of the body for it to be used there.

Like every cell, erythrocytes may be lysed under certain conditions, inparticular under osmotic pressure conditions In 1952, Teorrel introducedthe concept of erythrocyte membrane resealing, i.e., the reconstitutionof the erythrocyte membrane after lysis of the erythrocytes.

Thus, insofar as it was possible to open and close erythrocytes, itshould therefore be possible to introduce certain exogeneous componentsinto the erythrocytes. In addition to the basic technique whichcomprises introducing the erythrocytes into a solution contained in arecipient the osmotic pressure of which is successively changed toeffect lysis, then resealing, some other processes should be mentionedwhich effect lysis in an iso-osmotic medium by applying an electric orthermal shock to the erythrocytes. In some cases, viruses or viralproteins have also been used to perform this encapsulation.

Encapsulations have also been performed by dialysis in a cellophane bag.All the processes which have previously been carried out have the commondisadvantage of producing small encapsulation yields, of being extremelychancy with respect to their reproducibility and of only allowing thetreatment of small quantities of erythrocyte suspensions.

Thus, although encapsulation has been reported of some enzymes, of theIX factor, of desferrioxamine, of organic acids, of glucose, ofsaccharose, of the A fragment of the diphtheric anti-toxin and ofmethothrexate, these processes have remained at the laboratory stage andit is still impossible for them to be applied to man.

It is only a recent publication by Green R. et al. (The Lancet (1980),Aug. 16th, 327-330) which describes the Desferal® (Ciba-Geigy)encapsulation and the use of the resulting erythrocytes in man.

However, having been produced by simple dilution in a hypo-osmosticmedium, this encapsulation provides very low incorporation yields ofDesferal in the erythrocytes.

For this reason, the object of the present invention is to perfect aprocess and an apparatus which allow the very high yield encapsulationin erythrocytes of substances which have a biological activity, andallow large quantities of erythrocyte suspensions to be treated whileensuring the maintenance of good sterility conditions which permit theuse of erythrocytes thus obtained in man or animals.

Although the present invention has been developed for the encapsulationof a large number of compounds having a biological acitivity, one of theclasses of compounds of a biological activity appears to be of moreparticular interest. This class embraces allosteric effectors ofhaemoglobin.

At the stage of the lungs, haemoglobin forms with oxygen a reversibleaddition product: oxyhaemoglobin.

This addition product decomposes at the level of the capillary system torelease oxygen, because the partial pressure of oxygen in the capillarysystem is lower (about 70 mm of Hg) than in the lungs (about 100 mm ofHg).

Under normal conditions, only some, about 25% of the haemoglobindissociates, and the remainder returns to the lungs through the venoussystem.

In vitro, it has been possible to show the function of some substances,such as inositol hexaphosphate (IHP), and pyridoxal phosphate forexample, which are capable of combining with haemoglobin at the bindingsite of 2,3-diphosphoglycerate.

These products which are termed "allosteric effectors of haemoglobin"(AEH) modify the allosteric conformation of haemoglobin, the effect ofwhich is to reduce the affinity of haemoglobin for oxygen.

A reduction in the affinity of haemoglobin for oxygen facilitates therelease of oxygen from oxyhaemoglobin, even for relatively elevatedoxygen partial pressures.

Under these conditions, without the total oxygen content of the bloodbeing notably modified, it is possible to envisage increasing the oxygenrelease capacity of the blood to the tissues by using red blood cellscontaining a modified haemoglobin.

This type of treatment of haemoglobin permits an improved oxygenation ofthe tissue by improving the release of oxygen.

The uses of these allosteric effectors of haemoglobin are potentiallynumerous, for example in the treatment of certain situations of hypoxy.

They may improve or accelerate certain therapeutic processes, forexample in the prevention of heart infarction due to ischaemia, or theycan modify the respiratory physiology at increased altitudes or lowdepth.

Of course, the essential problem is to bring the effectors in situ sothat they may become attached to the intracellular haemoglobin.

Attempts at transforming haemoglobin in solution by allosteric effectorsof haemoglobin have been carried out in vitro, but practicalapplications are still incapable of a therapeutic use due to theinadequate life span of haemoglobin in solution and to the reducedproperties which it then has in transporting oxygen.

U.S. Pat. No. 4,192,869 and European Patent No. 0,001,104 describeprocesses which transform red blood cells by an interaction of saidblood cells with liposomes loaded with allosteric effectors ofhaemoglobin, in particular IHP (inositol hexaphosphate).

SUMMARY OF THE INVENTION

The process and apparatus according to the present invention allow thetransformation of haemoglobin under conditions which allow its use on atherapeutic level, using an allosteric effector of haemoglobin as acompound having a biological activity.

In order to achieve this, the present invention proposes a process forthe encapsulation in human or animal erythrocytes of at least onesubstance having a biological activity, characterised in that theprimary compartment of at least one dialysis element is continuouslysupplied with an aqueous suspension of erythrocytes, the secondarycompartment of this dialysis element contains an aqueous solution whichis hypotonic with respect to the erythrocyte suspension in order to lysethe erythrocytes, and in that the erythrocyte lysate is then in contactwith said substance of a biological activity, and in that, in order toreseal the membrane of the erythrocytes, the osmotic and/or oncoticpressure of the erythrocyte lysate is increased after it has beenbrought into contact with said substance having a biological activity,and in that the suspension of resealed erythrocytes is recovered.

Within the context of the present invention, the term "dialysis element"will generally be understood as an element comprising two compartmentswhich are separated by a dialysis membrane through which an ionicexchange may take place which allows the osmotic pressure of an aqueoussolution in one of the compartments to be modified in a controlledmanner by introducing an aqueous solution of a salt into the othercompartment. This type of dialysis element is widely used as much in themedical field for the operation of continuous flux haemodialysis, forexample peritoneal dialysis or plasmatic exchange with separation of thecells or the plasma, as in the industrial field in purificationoperations, for example in the pharmaceutical and food industries.

It should be understood that the present invention is not concerned withthe structure of such a dialysis element which may be of any design andmay comprise, for example hollow fibres or membranes with any passagesthrough several dialysis chambers, the essential aspect of the processbeing that it is possible to modify the tonicity of the aqueous solutionwhich is introduced into the primary compartment, due to the adjoiningin the secondary compartment of an aqueous solution which is hypotonicwith respect to the suspension of erythrocytes, the purpose of this, ofcourse, being to lyse the erythrocytes.

The erythrocyte lysate must be in contact with the substance having abiological activity which is to be encapsulated, but it is possible tointroduce this substance either before, or during lysis or even possiblyafter lysis, but in the latter case, the encapsulation yields ar not asgood.

In fact, in the practical application of the process for encapsulatingbiologically active substances in erythrocytes, two situations may beencountered:

(a) The size of the substance to be encapsulated exceeds the porediameter of the membrane separating the primary and secondarycompartments of the dialyzer. This is the case with a macromolecularsubstance. Under these conditions, there is no loss of that substanceduring dialysis leading up to the phase of introduction of the substanceinto the erythrocytes and the final yield will correspond to theequilibrium phase obtained after lysis of the erythrocytes.

(b) The substance to be encapsulated is of reduced size (below 10,000daltons) and, accordingly, will dialyze in the secondary compartment ofthe dialyzer during the lysis phase. This will result in a reduction inthe yield of encapsulation of the substance in the erythrocytes. Toreduce this loss of substance, which is highly undesirable in the caseof expensive or rare compounds, the process may be modified as follows:

In effect, the erythrocytes are initially washed in a medium containingsolutes of which the osmotic pressure is of the order of 220 to 300 mos.The lysis of these erythrocytes only takes place below 200 mos. It isduring this phase of reduction of the osmotic pressure to 200 mos thatthe main loss of the substance to be encapsulated occurs if it has beenintroduced before the beginning of dialysis. To reduce this loss bydiffusion, it is thus sufficient to introduce the substance into themedium at the beginning of lysis of the erythrocytes, i.e. when theionic strength of the suspension has been adjusted to between 180 and220 mos.

In this variant, before introducing the erythrocytes suspension in thefirst dialysis element, the ionic strength of the suspension is lowereduntil 180-220 mos and it is only when the osmotic strength reaches thecorrect value that the substance to be encapsulated is introduced. Theso obtained suspension is then treated as described herein before.

Preferably, to this end, an aqueous erythrocyte suspension iscontinuously fed to the primary compartment of an additional dialysiselement of which the secondary compartment is fed with an aqueoussolution that is hypotonic in relation to the erythrocyte suspension inorder to adjust the ionic strength of the primary compartment to between180 and 220 mos.

There are several possibilities of increasing the osmotic pressure ofthe erythrocyte lysate when the membrane of the erythrocytes is to beresealed.

In a first process, the osmotic pressure of the erythrocyte lysate isincreased by passing it into the primary circuit of a dialysis element,the secondary circuit of which contains a hypertonic solution withrespect to the lysate, the solution being continuously recovered afterresealing.

In a second process, the osmotic pressure of the lysate is increased bymixing it with a hypertonic and/or hyper-oncotic solution with respectto said lysate.

It should be pointed out that the product which is treated is an aqueoussuspension of erythrocytes, i.e., in most cases, it is preferable towork on a "synthetic" erythrocyte suspension and not on the completeblood, i.e., a suspension which mainly contains only erythrocytes as theoriginal blood component.

This is why, in addition to the dialysis-resealing phases, the processmay preponderantly comprise the following phases:

washing and preparing the erythrocytes for lysis and resealing,

washing and, optionally, resuspending the transformed erythrocytes inplasma or in an artificial composition.

Numerous known methods and variants may be added to the apparatus forencapsulating substances in red cells by the process according to theinvention. The methods in question are essentially:

centrifuging to separate the cells from the plasma and for washing;

separation by means of continuous-flow or semicontinuous-flow cellseparators;

using continuous-flow and semicontinuous-flow devices for separating thecells on semipermeable membranes by known methods of plasma phaeresis onmembranes or hollow fibers;

washing by the same methods.

It is also well known in connection with blood transfusion that it isoccasionally desirable to eliminate the white cells or the plateletsbefore transfusion for immunological reasons or for reasons associatedwith the formation of microaggregates.

The same situations may be encountered in the use of lysed and resealedred globules which have encapsulated a substance of biological interest.Accordingly, it would be possible to introduce into the process anelement for separating leucocytes, such as a conventional absorptionfilter. The other conventional processes for separating leucocytes mayobviously be adapted to that element.

The use of a "synthetic" erythrocyte suspension allows the osmoticpressure of said aqueous suspension to be controlled accurately and thusensures a good reproducibility of the results which are obtained.

When this aqueous erythrocyte suspension is being prepared, it ispossible to carry out various types of "conditioning" of the suspension.

First of all, it is possible to introduce the erythrocytes into aniso-osmotic medium with respect to the blood, i.e., under naturalconditions of osmotic pressure, and under these conditions, lysis willbe carried out in the presence of an aqueous solution which is hypotonicwith respect to the suspension of erythrocytes.

However, it is also possible to condition the erythrocytes in an aqueoussolution containing substances which may diffuse through the membrane,and under these conditions, the erythrocytes will tend to swell and itwill be possible to carry out lysis using an iso-osmotic medium or even,in some cases, a hyper-osmotic medium (these pressures being evaluatedwith respect to the normal blood osmotic pressure).

It may also be advantageous during the preparation of the syntheticerythrocyte suspension which is to be treated to add to this suspensionmacromolecular compounds, in particular colloids, in order to produce inthis solution an oncotic pressure, and the effect of this pressureduring lysis will be to restrict the loss of intracytoplasmic moleculesand, in particular, the loss of haemoglobin.

Of course, it is possible to envisage, at the stage of lysis orresealing, the use not only of modifications in the osmotic pressure ofthe solution, but also modifications in the oncotic pressure in order toimprove lysis or resealing.

A major advantage of the process according to the present invention, inaddition to the possibilities of controlling the sterility and theabsence of pyrogen which allows the use in man of products obtained, isthat this process lends itself particularly well to a regulation bycontrolling the parameters, such as notably time, volume, temperature,flow rate, exchange area, conductivity and ionic strength.

The different operational parameters of the process may vary dependingon the nature of the treated elements, but lysis will preferably becarried out at a temperature between 0° and 10° C. and resealing at atemperature between 20° and 40° C.

Analysis of the phenomenon of erythrocyte resealing shows thatconditions different from those described in the foregoing may well beinvolved:

introduction of the substance to be encapsulated into the suspension oflyzed erythrocytes at 2° to 4° C. and not before entry into the dialysiscircuit;

stages at different temperatures and at successive levels, leadinggenerally to a temperature gradient of 2° to 6° C. at the beginning tobetween 35° and 42° C. between lysis and resealing;

introduction of resealing solutions of substances at different points ofthe above-mentioned levels;

variable compositions of the resealing solutions or buffers.

Finally, the encapsulation in low concentrations of highly activesubstances which are protein-like in character or which are readilyadsorbed onto the plastic surfaces used in the process makes it appearadvisable to pretreat the dialysis circuit in certain cases. Theseabsorption phenomena are well known and, in view of the large surfacesused, may lead to the loss of a very large quantity of the substance tobe encapsulated in the process. This might be the case, for example,with substances such as cytokines or enzymes. Various methods may beused for modifying the surfaces of the transformation circuits, such asfor example the preliminary use of a coating based on a protein, such asalbumin.

Although these methodological precautions are not directly associatedwith the process, they can perform a major role in its application.

As previously indicated, it has been found that although the processaccording to the present invention is a continuous process, yields areobtained which are widely superior to the yields observed by the saidtechnique of a "dialysis bag". Moreover, it is possible to treatconsiderable volumes of blood, the treatment being carried out veryrapidly.

The differences which have been observed between these results may bepartly explained by the fact that the two processes are fundamentallyvery different. In fact, one of the processes is a kinetic method,whereas the other is an equilibrium method. Under these conditions, theconsiderable shortening of the period during which the red blood cellsare maintained in a lysed form restricts the physicochemicalmembrane-medium exchanges accompanied by a loss of constituents of theerythrocyte membrane, greatly reducing the proportion of red blood cellsfor which lysis has become irreversible.

Of course, in addition to the advantages associated with the rapidity oftreatment which can be obtained by the process, it should be mentionedthat it is possible, for the dialysis phase, to treat large bloodvolumes, for example 200 ml of washed packed cells over a period of 10minutes, instead of 10 to 20 ml over a period of 21/2 hours by themethod of a dialysis bag.

The process according to the present invention may be carried out toencapsulate a large number of substances having a biological activity.In particular, these substances are as follows:

proteins,

enzymes,

hormones,

immunomodulators,

substances generally having a pharmacological activity,

substances modifying the metabolism of erythrocytes, such as, forexample allosteric effectors of haemoglobin, and

protective substances, notably cryoprotective substances of haemoglobinand erythrocytes.

nucleic acids.

As explained above, allosteric effectors of haemoglobin should bementioned from among the substances which have a biological activity andwhich may be used in the present process.

Among the allosteric effectors of haemoglobin, inositol hexaphosphate(IHP) should be mentioned, but other effectors may be used, for examplesugar phosphates, such as inositol pentaphosphate, inositoltetraphosphate, inositol triphosphate, inositol diphosphate anddiphosphatidinylinositol diphosphate.

It is also possible to use polyphosphates, such as nucleotidetriphosphates, nucleotide diphosphates, nucleotide monophosphates,alcohol phosphate esters and pyridoxal phosphate.

A whole series of natural or synthetic substances of increasing interestin cell or molecular therapy may be encapsulated, namely:

prostaglandines,

leukotrienes,

cytokinins or synthetic immunomodulators (immunoactivators ofimmunosuppressors). In this particular category, it may be advisable toconsider using the process for modulating the immuno-defence system ofthe organism by means of natural or synthetic substances which can bedirected by the process:

onto the reticuloendothelial system, the preferential site whereerythrocytes disappear during the ageing process;

onto certain cells, such as monocytes, which are intended to betransformed into tissular macrophages, in particular by means of anantibody dependent cytolysis reaction (ADCC);

onto the lymphatic system which represents the preferential site ofelimination of transfused erythrocytes, for example by theintraperitoneal route.

At the present time, the most interesting compounds are:

α, β or γ interferons or genetic recombination interferons;

interleukine II;

synthetic immunomodulators, particularly derivatives of muramyldipeptide (MDP).

For the same reasons which are behind the selective and effectivetargeting of the natural or synthetic immunomodulators, apart from aretarding effect and the limitation of toxicity, it may be very usefuland effective to utilize the process for encapsulating, targeting andactivating substances which show anticarcinogenic properties.

Of the many other natural substances, particularly enzymes, which may beencapsulated by the process according to the invention, it is possibleto give numerous examples.

Three particularly interesting examples are mentioned in the following:

(a) Encapsulation of enzymes from the metabolism of glucose

Glucose is a substrate which diffuses rapidly and freely through themembrane of the red cell.

The encapsulation of enzymes from the metabolism of glucose,particularly hexakinase, would make it possible to act on theextraerythrocytic metabolism of glucose and, hence, to modulate theconcentration of blood glucose other than by the action of hormones,such as insulin.

The intraerythrocytic stability might necessitate the addition ofprotease inhibitors, as in the case of insulin, or other enzymeinhibitors.

(b) Encapsulation of carbonic anhydrase

The metabolism of CO₂ presents serious problems in the reanimation ofcertain patients showing a high overload of blood CO₂ which, hitherto,has been poorly controlled.

The encapsulation of carbonic anhydrase in more than the naturally highconcentrations in the red cell would enable the equilibrium:

    CO.sub.2 →CO.sub.3 H.sup.-

to be displaced, the latter compound being able to be eliminated bydialysis of the blood of the patient.

(c) Encapsulation of enzymes from the metabolism of histamine

We have shown that histamine diffuses through the erythocytic membranein a manner similar to glucose, but kinetically more slowly.

This fundamental point enables the process to be used for encapsulatingenzymes from the metabolism of histamine, more particularlydiamino-oxidase (histaminase) and methyl-histamine transferase, whichare the two preferential enzymes from the catabolism of histamine.

By virtue of this particular point, the process according to theinvention may be considered for possible adaptation for the treatment ofhistamine overloads, particularly chronic histamine overloads, inallergic patients.

Without being restrictive in any way, these examples illustrate theencapsulation of enzymes capable of modifying the metabolism of certainpatients suffering from acquired or congenital anomalies and theencapsulation of detoxification enzymes.

According to the invention, it is possible to target the activesubstance towards the macrophages, so it is very interesting to use asactive substance a derivative or analog of MDP since it is known thatsuch compound has an activity through the macrophages (C. Leclerc et L.Chedid, "Macrophage activity by synthetic muramyl peptide inlymphokines", E. Pick Ed. Academic Press Inc. 7, 1-21, 1982).

The following French patents are cited as reference for analog orderivative of MDP:

74 22909 dated 1.07.74

75 28705 dated 18.09.75

75 29624 dated 26.09.75

76 06820 dated 10.03.76

77 02646 dated 31.01.77

76 19236 dated 16.07.76

78 16793 dated 5.06.78.

Among these products, the MDP derivatives wherein the peptidic chain ismono- or di-esterified on the carboxylic group of D-glutamic acid withC₁ -C₁₀ -alkyl chains preferably C₁ -C₆ -alkyl, especially the butylicester of D-glutamine, are interesting.

The mono- and di-esters having in α a C₄ -C₁₀ -alkyl chain, especially 4carbon atoms, and in γ a lower C₁ -C₃ -alkyl chain are also especiallyinteresting.

Of course, it is possible, if necessary and/or useful, to encapsulatesimultaneously several compounds having a biological activity.

For some of these products, encapsulation allows a selective targetingon certain organs or the induction of a prolonged delay effect which mayamount to from 15 to 25 days or more, according to the present dataobtained in animal.

It is equally important to note that the encapsulation may induce aprotective effect for some drugs which are toxic with respect to most ofthe tissues and organs for which the drug is not intended. the same typeof activity is observed in the induction of a protective effect withrespect to the drug or to the transported hormone which, in some cases,are capable of inducing an immunizing response, or when they areinjected into individuals who have an antibody against these substancesin the blood circulation for example an antiinsuline, antibody. Thisprotective effect may also be exercised with respect to a metabolicdeterioration in the drug or hormone.

It may be necessary to add protease inhibitor to enhance the stabilityof the substance in the resealed cells, such as in the case of insulineor other enzymatic inhibitors.

Finally, the process makes it possible to introduce compounds whichprotect haemoglobin and the membrane, enabling in particular theintegrity of the erythrocytes to be protected.

The compounds in question may be cryoprotectors or lyoprotectors, thelatter being particularly effective in cases of freeze-drying.

Finally, it should be noted that the erythrocytes may be frozen in thelyzed state and may be resealed on defreezing or, in cases offreeze-drying, during the re-hydration phases. This latter situationmakes it possible theoretically to resolve the passage of water throughthe membrane during re-hydration.

The present invention also relates to an apparatus intended for theencapsulation in human or animal erythrocytes of at least one substancehaving a biological activity, characterised in that it comprises atleast the following combined elements:

a dialysis element comprising at least one primary compartment and onesecondary compartment which are separated by a dialysis membrane,

a device for the continuous supply of the erythrocyte suspension intothe primary compartment of said dialysis element,

a device which allows an aqueous solution to be introduced into thesecondary compartment of the dialysis element,

a device ensuring the introduction of the substance having a biologicalactivity into the primary compartment of the dialysis element, and

a device ensuring the transfer of the effluent solution from the primarycompartment into a resealing assembly, comprising at least one means forincreasing the osmotic and/or oncotic pressure of the effluent solution.

The structure of the dialysis element is not a characteristic of theapparatus according to the present invention, and it is possible to useany type of dialysis element, as previously indicated, within the scopeof the process.

Any device, notably a pump system, and in particular peristaltic pumpswhich are presently used within the dialysis field may be envisaged as acontinuous supply device.

The apparatus according to the present invention may include differentmodes of production, in particular with respect to the resealingassembly.

The first mode of production of the resealing assembly may be composedof a dialysis element comprising at least one primary compartment andone secondary compartment which are separated by a dialysis membrane,the effluent solution being introduced into the primary compartment bythe transfer device and the device for increasing the osmotic pressurecomprising a solution which is hypertonic with respect to the effluentsolution, such hypertonic solution being contained in the secondarycompartment of the dialysis element.

However, it is also possible to provide other productions of thisresealing assembly which may be composed of an enclosure comprising adevice for introducing a solution which is hypertonic with respect tothe effluent solution.

Since lysis is preferably carried out at a fairly low temperature,between 0° and 10° C., and preferably in the region of 4° C., and sinceresealing is preferaly carried out at a higher temperature, for examplebetween 20° and 40° C., the apparatus according to the present inventionwill preferably comprise a device for heating the suspension to betreated. This heating device may be located between the dialysis elementand the resealing assembly, or directly inside the resealing assembly.

In another production, it is possible to provide after the firstdialysis element a receiver which recovers all of the volume ofsuspension to be treated, then, after having changed the tonicity of theaqueous solution circulating in the secondary compartment of thedialysis element, it recycles the erythrocyte lysate thus obtained inthe same dialysis element, and in this case, this dialysis element isused in a first step for lysis of the erythrocyte suspension, and in asecond step as the resealing assembly.

The apparatus according to the present invention may also embody controlcircuits which, in response to modifications in characteristics of thetreated suspension, will act on all of the parameters of the reaction inorder to restore the characteristics of the suspension to the level of apredetermined order.

BRIEF DESCRIPTION OF THE DRAWINGS

Other characteristics and advantages of the process and the apparatusaccording to the present invention will be revealed in the followingdescription of the Figures:

FIG. 1 shows a first embodiment of the apparatus according to theinvention.

FIG. 2 shows a second embodiment of the apparatus according to theinvention.

FIG. 3 shows a third embodiment of the apparatus according to theinvention.

FIG. 4 shows a fourth embodiment of the apparatus according to theinvention.

FIG. 5 shows the partial oxygen pressure as a function of the percentageof saturation of the erythrocytes.

FIG. 6 is an electron microscope photograph of a resealed erythrocytesuspension.

FIG. 7 shows the percentage haemolysis as a function of the osmoticpressure for various prewashes.

FIG. 8 shows the gross and net yields as a function of blood throughputfor various prewashes.

FIG. 9 shows the quantity of desferal encapsulated as a function of theinitial concentration of the product.

FIG. 10 shows the effect of the resealing temperature.

FIG. 11 shows the variation in the p₅₀ for various concentrations ofIHP.

FIG. 12 shows the variations in various blood oxygen pressures.

FIG. 13 shows the trend of cardiac output in dependence upon thevariation in the p₅₀.

FIG. 14 is a microphotograph of control and resealed residues.

DETAILED DESCRIPTION OF THE INVENTION

The apparatus of FIG. 1 is composed of a dialysis element 1 comprising aprimary compartment 2 and a secondary compartment 3 which are separatedby a dialysis membrane 4.

The primary compartment 2 is supplied with an erythrocyte suspensioncontained in a reservoir 5 positioned in an incubator 6.

Before being introduced into the dialyser 1, the blood is brought to thelysis temperature which is generally 4° C., by means of a heat exchanger7.

The erythrocyte suspension is circulated by means of peristaltic pumpssuch as 8.

In the production which is being described, the compound having abiological activity is introduced into the circuit by means of aperistaltic pump 9 before the erythrocyte suspension penetrates into theheat exchanger 7.

The secondary compartment 3 of the dialysis element is supplied with ahypotonic solution contained in a receiver 10 which is positioned in anincubator 11. This hypotonic solution is introduced into the secondarycompartment of the dialysis element by means of a peristaltic pump 12and through a heat exchanger 13. After passing into the secondarycompartment of the dialysis element, the hypotonic solution returns tothe receiver 10. It may also be discarded.

In order to ensure that lysis of the erythrocytes is complete, it ispossible, as illustrated in FIG. 1, to position at the outlet of thedialysis element 1 a delay line 14, i.e., usually a simple long tubing,for example.

In the most frequent case when the resealing operation has to be carriedout at a temperature above the lysis temperature, a heat exchanger, suchas 15 is introduced after the delay line, which exchanger will allow thelysed erythrocyte suspension to be brought to a temperature of, forexample 37° C.

The lysate is continuously introduced into a receiver 16 which iscontained in an incubator 17. The resealing solution which is introducedinto a receiver 18 contained in an incubator 19 is added continuously,or all at once into this receiver 16 by means of a peristaltic pump 20.

When the resealing operation is complete, the erythrocyte suspension inwhich the compound having a biological activity is encapsulated isdirectly removed from the receiver 16.

One of the advantages of the apparatus according to the presentinvention is that it is possible to permanently control all theparameters of the reaction.

Of course, it is possible to control the temperature of the differentcirculating fluids by means of devices which have not been shown becausethey are well known from the prior art.

However, it is very advantageous to be able to control the pressure ofthe fluids and also their osmotic pressure in order to ensure that theexchanges will be carried out under optimum conditions.

Of course, it is unnecessary to go into details about these controldevices which are known. At least one manometer such as 21 willpreferably be provided to control the pressure of the fluids in theprimary circuit, and an element for measuring the osmotic pressure, forexample by measuring the conductivity using a device such as 22, theseelements acting to modify the other parameters of the reaction, such asthe feed flow rate, in order to restore the values of the pressure andthe osmotic pressure to the ordered values.

It will be the same on the secondary circuit where a device denoted withreference numeral 23 will allow the pressure of the fluid as well as theosmotic pressure to be measured in order to maintain the characteristicsof the fluids at certain set values.

Other control devices, such as 24 and 25 are also illustrated whichensure identical functions to those previously defined.

FIG. 2 illustrates an apparatus which is identical to that of FIG. 1 upto the level of the second thermal exchanger denoted with numeral 15 inFIG. 1, and since this part of the apparatus is absolutely identical tothat of FIG. 1, it will not be described again.

On the other hand, the resealing stage is carried out by means of asecond dialysis element 30 comprising a primary compartment 31 and asecondary compartment 32 which are separated by a dialysis membrane 34.

A hypertonic resealing solution circulates in the secondary compartment32 by means of a peristaltic pump 35 which draws out this hypertonicsolution in the receiver 36 and guides it by means of a heat exchanger37 into the secondary compartment of the dialysis element. Thishypertonic solution may be recycled in the receiver 36.

The lysis suspension originating from the first dialysis element, andafter passing into the delay line and into the heat exchanger, isintroduced into the primary compartment of the dialysis element 30, inwhich the erythrocytes are resealed, the suspension being recovered in areceiver 38 contained in an incubator, after resealing.

Here again, as previously mentioned, different control devices, such as40, 41 and 42 are provided which allow a continuous control of theoperational parameters of the process.

The flow rate of the primary circuit containing the erythrocytes isgenerally between 20 and 80 ml/min, whereas the flow rates of thehypertonic or hypotonic solutions of the secondary circuit are betweenabout 100 and 1 000 ml/min.

In the tests which will be described in the following, the areas ofdialysers which are used are generally about 0.41 m².

The pressures in the circuits approach 100 mm of mercury depending onthe flow rates which are used.

The ionic strength of the lysis solutions is about 40 mosmoles and theionic strength of the resealing solutions is about 300 mosmoles.

The diagram of FIG. 2 which is symmetrical for the two operations oflysis and resealing shows that it is possible to use an apparatus whichonly comprises a single dialysis element, and that it is possible tomake it operate during a first period to lyse the erythrocyte suspensionthen, after having recovered all of the lysed solution, to make thisdialysis system operate for the resealing operation by changing only thetonicity of the solution being supplied to the secondary compartment.

In the FIG. 3, the osmotic pressure of the erythrocyte suspension may beadjusted to 200 mos in order to limit the loss of substance to beencapsulated, as mentioned in the foregoing.

The size of the dialysis element 1 is selected in such a way that,taking the throughputs and volumes into account, the reduction in theionic strength of the erythrocyte suspension brings the suspension toapproximately 200 mos. The substance to be encapsulated which isaccommodated in the container 50 is introduced by means of the pump 51into the output circuit of the dialyzer 1 which may be formed simply bya conventional continuous throughput pressure syringe.

The erythrocyte suspension coming from the dialyzer 1 thus continuouslyreceives the substance to be encapsulated and enters the primary circuit27 of a dialysis element 28 of which the membrane 26 has a surface areaselected in such a way that the erythrocytes entering the primarycircuit undergo lysis by the initial process.

The secondary circuits 3 and 29 may be fed in series with the samehypotonic solution as shown in FIG. 1.

The ratio between the surface areas of the membranes 1 and 26 isselected to optimize the molecular exchanges in the two phases of thedialysis process.

The dialysis elements 1 and 28 may either be separated to enableexisting commercial elements to be used or, alternatively, may beintegrated in a two-compartment dialyzer specially adapted to theprocess, which may readily be achieved by means of present technology.

The outlet of the dialyzer 28 returns to the elements of the basicdiagram shown in FIG. 1.

FIG. 4 shows one particular embodiment of the process according to theinvention.

The conditions described hitherto are particularly suitable for theencapsulation of a substance in a total volume of approximately 200 to400 ml or for the use of a continuous-flow transformation. By modifyingthe surface areas of the dialyzers and the throughputs, variable volumesmay be treated.

However, it is useful to describe in detail a continuous recyclingcircuit for the extreme conditions:

(a) Unless resealing is carried out continuously, the transformation ofa volume of an erythrocyte suspension amounting to several litersintroduces a significant difference into the kinetics of the phenomenonbetween the first and last erythrocytes to pass through the phases ofthe dialysis process.

(b) Similarly, the handling by means of a minidialyzer of smallquantities of blood (a few ml) for the encapsulation of highly activesubstances which do not require large volumes of blood for the doses tobe administered introduces an additional parameter. This is because theeffectiveness of a dialyzer depends upon its surface area and,accordingly, allows a predetermined dialysis equilibrium, although thisequilibrium is only reached according to kinetics which are slower, thesmaller the surface area. Accordingly, simple adaptation of throughputto surface area is not sufficient for obtaining encapsulation undersuitable conditions.

In this embodiment, the surface of the dialyzer 1 is reduced and thethroughput of blood through the primary circuit increased in such a waythat, at the outlet end of the dialyzer 1, the osmotic pressure is onlyreduced for a fraction of what would be necessary for causing lysis ofthe red cells. The erythrocyte suspension is continuously recycled intothe initial reservoir 5. The heat exchanger 15 may be programmed merelyon completion of transformation. The same applies to the pump 20.

This apparatus enables the process to be carried out by successive andprogressive modifications of the ionic strengths of the media whichenables the kinetics of the dialyzer 1 to be ignored through theintroduction of complementary kinetics associated with the volume ofblood to be treated and with the throughput through the circuit.

This modification makes it possible readily to adapt the process to avery wide range of volumes of blood to be treated, taking into accountthe technical characteristics of currently available dialyzers, andhence, in principle, to establish the optimal encapsulation conditionsfor very different conditions. It is thus possible to transform bothlarge volumes of blood and also volumes of a few ml intended for animalexperimentation or for the encapsulation of a rare and expensivesubstance.

The apparatus which are illustrated in FIGS. 1 and 2 have been used tocarry out the process which is claimed.

EXAMPLE 1 Incorporation of Desferrioxamine (Desferal)

In this Example, the apparatus which is illustrated in FIG. 1 is used.

The dialyser 1 has an area of 0.41 m² and is supplied with anerythrocyte suspension at a flow rate of 60 ml/min in the primarycompartment, while a lysis solution containing NaH₂ PO₄ -Na₂ HPO₄, pH7.4, 10 mM, CaCl₂ 0.5 mM and 2 mM of glucose is introduced into thesecondary compartment at a flow rate of 500 ml/min.

The erythrocyte suspension was obtained in the following manner: 5 ml ofsolution containing 250 mg of desferal are added to 200 ml of washedpacked red blood cells, having 70% hematocrit. Lysis is carried out at4° C. keeping the lysis suspension in a receiver 16 at this temperaturefor 10 minutes. 20 ml of NaCl 1.5M, containing 10% of PEG 4 000 are thenintroduced. The suspension is maintained in contact with this resealingsolution for 30 minutes at 37° C. All of the suspension is then removedand three washing operations are carried out using NaCl at 9 g/l.

It is useful to carry out an intermediate washing operation with asolution of about 200 mos. or 250 mos. to remove some erythrocytepopulations which have become fragile. Likewise, the resuspension in theinitial plasma or in regeneration solutions may be necessary for goodpreservation of the red blood cells.

A determination shows that 148 mg of desferal (that is a gross yield of59%) have been incorporated in the packed red blood cells, but it shouldbe noted that 12 mg of desferal were removed in the dialysis solution,which in fact results in a net yield of desferal incorporation in theerythrocytes of 63.5%.

If the supply rate of erythrocyte suspension is modified, bringing it to80 ml/min., the gross incorporation yield is 47.3% and the net yield is57.9%.

With a flow rate of 40 ml/min., the gross yield is 49.4% and the netyield is 59.1%.

With a flow rate of 20 ml/min., the gross yield is 46.1% and the netyield is 64.2%.

The gross yield is calculated upon the total quantity of the product andthe net yield is calculated upon the remaining product after the lysis.

These results show that there is an optimum flow rate of erythrocytesuspension for a given dialyser area, when the substance which is to beincorporated is a substance which is capable of diffusing through thedialyser membrane.

EXAMPLE 2 Incorporation of desferrioxamine (Desferal)

In this Example, the apparatus which is described in FIG. 2 is used,i.e., resealing is effected by means of a second dialysis at 22° C.using a dialysis element, such as 30.

In this case, the following resealing buffer is used: NaH₂ PO₄ --Na₂HPO₄ 10 mM, pH 7.4, CaCl₂ 0.5 mM, glucose 2 mM, NaCl 144 mM.

The other parameters are the same, and the erythrocyte suspension flowrate is 50 ml/min.

In this case, a net incorporation yield of 46.7% is observed, while thegross yield is 32.5%.

These results are easily explained, since desferal is a diffusiblesubstance and another new quantity of desferal tends to be lost duringthe second dialysis.

EXAMPLE 3 Incorporation of Insulin

In this Example, insulin is encapsulated in erythrocytes by using theapparatus illustrated in FIG. 1. Only the parameters which differ fromthose indicated in Example 1 will be mentioned in the following.

In order to allow the insulin to be determined, the product which isused is denoted by I¹²⁵ by the chloramine T method.

10 ml of insulin (4 U/ml-432.800 cpm/ml) are added to 200 ml of packedred blood cells which have been washed three times with NaCl at 9 g/l.

The erythrocyte suspension flow rate used is 60 ml/min. After lysis,incubation is carried out for 10 minutes at 4° C.

Resealing is effected at 37° C. using 20 ml of NaCl 1.5M containing 10%of PEG 4 000.

Incubation is carried for 30 minutes at 37° C. and the red blood cellsare then washed three times with NaCl at 9 g/l.

Under these conditions, the incorporation yield of the radioactivity is48% (±4%). Of course, this incorporation could not foresee thebiological activity of the insulin encapsulated in the red blood cells.It is well known that this hormone is modified in the presence of redblood cell haemolysate.

However, this result confirms the possibilities of incorporating aprotein of 7 000 daltons in erythrocytes.

In order to preserve the functional integrity of the insulin, it may benecessary to provide the incorporation of protective substances ofinsulin in the erythrocyte medium, and this may of course be easilycarried out using the apparatus according to the present invention.

EXAMPLE 4 Incorporation of Albumin

The process is carried out as described in Example 3, using albuminlabelled by I¹²⁵. Under these conditions, a yield of about 35% isobserved, and this result shows the influence of the molecular mass onthe incorporation yield of a protein.

The following Examples are to illustrate the encapsulation of allostericeffectors of haemoglobin, notably inositol hexaphosphate (IHP) by theprocess of the present invention.

EXAMPLE 5 Incorporation of IHP

In order to produce a more or less considerable transformation of redblood cells using inositol hexaphosphate, two types of solution, forexample are used which allow the ratio of IHP:Na to be varied.

Solution A

15 g of IHP 12 Na (12% H₂ O) in 55 ml of distilled water are passed overa 400 mesh 50 W DOWEX column equilibrated in H⁺ ions. 63 ml of acid IHPare obtained (14.2×10⁻³ mols).

This acid IHP is used to neutralize another solution of IHP 12 Na to pH7.4. 38.1×10⁻³ mols of IHP 12 Na in 153 ml are neutralized by 97 ml ofacid IHP containing 17×10⁻³ mols of IHP.

In all, a solution containing 55×10⁻³ mols of IHP and 457×10⁻³ mols ofsodium in 250 ml is obtained. This solution is diluted to obtain amedium of 250 mosmoles/liter.

Solution B

11 g of IHP 12 Na (12% H₂ O) are neutralized by 40 ml of N HCl in 750 mlof H₂ O to pH 7.4. A medium of 230 mosmoles/liter is obtained.

In this Example, the apparatus which is illustrated in FIG. 1 is used.

The dialyser 1 has an area of 0.41 m² and is supplied with anerythrocyte suspension at a flow rate which varies, depending on thetests, from 20 to 60 ml/min.

The secondary circuit is supplied at a rate of 500 ml/min with alysis-buffer having the following composition:

CO₃ HNa 10 mM

PO₄ H₂ K/PO₄ HK₂ 10 mM,

glucose 2 mM, pH 7.4.

The erythrocyte suspension which is used was obtained in the followingmanner:

An equivalent volume of one of the solutions A or B is added to 200 mlof packed red blood cells which have been washed three times with asolution of NaCl at 9 g/l (70% hematocrit).

After centrifugation, the slightly hemolyzed supernatant is discarded.

Lysis is carried out at 4° C. The lysis suspension is kept in a receiver16 for 10 minutes at 37° C. in the presence of IHP.

The lysis suspension is resealed by adding 10% by volume of a solutioncontaining NaCl 1.5M, PEG 4 000 at 10%.

After incubating for 30 minutes at 37° C., the red cells are washedthree times with a solution of NaCl at 9 g/l and are resuspended in theoriginal plasma.

It is useful to carry out an intermediate washing operation with asolution of about 220 mos. in order to remove some erythrocytepopulations which have become fragile. Likewise, the resuspension in theinitial plasma or in regeneration solutions may be necessary for goodpreservation of the red blood cells.

The results of these tests are provided in the following Table.

    __________________________________________________________________________               Lysis NaCl                                                         Treatment                                                                           Control                                                                            resealed without IHP                                                                     Solution A                                                                             Solution B                                     __________________________________________________________________________    Flow rate  60         60 40 20 60 40 20                                       (ml/min)                                                                      P.sub.50                                                                            20.6 19.6       51.5                                                                             66.5                                                                             77.0                                                                             36.8                                                                             40.7                                                                             46.5                                     (mm Hg)                                                                       n of Hill                                                                           2.40 2.50       1.64                                                                             1.97                                                                             1.25                                                                             1.49                                                                             1.53                                                                             1.47                                     30-70%                                                                        (mm Hg).sup.-1                                                                __________________________________________________________________________

The oxygen binding curves shown in FIG. 5 are measured at 25° C. by therapid diffusion method using the apparatus of Duvelleroy (J. Appl.Physiol. 28, 227-233 (1970) and Tesseirre et al. (Bull. Physiopath.Resp. 11, 837-851 (1975).

The curves in FIG. 5 represent the oxygen partial pressure in mm ofmercury as a function of the saturation percent of the erythrocytes forvarious flow rates of erythrocyte suspensions and depending on thenature of the solution which is used, A or B.

The half-saturation pressure denoted P₅₀ in the Table is an indicationof the affinity of oxygen for haemoglobin. The slope of the curve in thevicinity of half-saturation, i.e., between 40 and 60% represents thedegree of co-operation of the four fixation sites of oxygen inhaemoglobin, and this slope allows the HILL coefficient to becalculated.

An improvement in the oxygen release is detected, i.e., a decrease inthe affinity of haemoglobin for oxygen, either by a reduction in theHILL coefficient, i.e., a reduction in the slope of the curve of FIG. 5in the vicinity of 50% of saturation, or by a complete displacement ofthe curve towards the right which corresponds to an increase in theoxygen partial pressure in the vicinity of 50% of saturation.

For the control erythrocytes of curve T, the P₅₀ of which is 19.3 mm ofmercury, it is observed that the same erythrocytes treated by theprocess of the Example in the initial presence of physiological serumhave a P₅₀ of 18.5 mm of Hg, whereas after incorporation of IHP by theprocess according to the present invention, the P₅₀ values which areobserved vary between 36.8 and 77 mm of Hg depending on the flow rateswhich are used and on the nature of the IHP solution used.

EXAMPLE 6

Using the solution A which is diluted to obtain 210 mosmoles/liter,lysis is carried out at 4° C. at a flow rate of 20 ml/min, followed by aresealing operation lasting for 30 minutes at different temperatures.The following Table shows the influence of the resealing temperature onthe P₅₀ value which is obtained, but also on the yield of reconstitutedred blood cells which is expressed by calculating the ratio of thehematocrits of the suspensions of reconstituted red blood cells to thehematocrit of the initial suspension for a total equivalent volume.

    ______________________________________                                                                   Yield                                              Resealing temperature                                                                         P.sub.50 (mm Hg)                                                                         (hematocrit)                                       ______________________________________                                        25° C.   42         27%                                                30° C.   48         36%                                                35° C.   50.5       45%                                                40° C.   54.5       50%                                                ______________________________________                                    

After resealing and maintaining in a plasmatic medium, most of the redblood cells assume a discocytic shape, although slightly microcytic. Themore or less vesicular forms are not significantly more numerous than inthe native population. This fact is confirmed by FIG. 6 which shows ascanning electron microscope photograph of a resealed erythrocytesuspension, the P₅₀ of which was 68 mm of Hg.

The experimental conditions which have been described show the greatpossibilities of use of the continuous flow dialysis to produce, undervariable conditions, the incorporation of IHP or other allostericeffectors of haemoglobin, in order to modify the oxygen-releasingproperties of blood.

The control of the parameters of the reaction of lysis-resealing,volumes, flow rates, temperatures and compositions of the media may beeasily carried out by means of the experimental apparatus used.

This apparatus allows the rapid transformation of large quantities ofred blood cells. It could possibly be incorporated in an extra-corporealcirculation circuit. The conditions of use allow a therapeuticapplication in man.

The method of incorporating allosteric effectors of haemoglobin in redblood cells which is described in this patent provides a number ofobvious advantages over the liposomal method described in U.S. Pat. No.4,192,869;

(1) The method does not use exogenous phospholipids and cholesterol andthereby greatly reduces the problems of toxicity and immunologicalactivity which have been acquired by the red blood cells.

(2) It allows a very accurate quantitation of the concentration ofallosteric effectors of haemoglobin which is incorporated andconsequently allows the obtention of the desired P₅₀ value.

(3) The incorporation process is faster and may be widely automated.

(4) The reproducibility of this method is greater than that of theliposomal method.

The following Examples carried out with Desferal using the method ofExample 1 demonstrate the effect of various parameters of the process.

EXAMPLE 7 Influence of Prewashing on the Lysis Curve

As already mentioned, it is possible to swell the erythrocytes beforelysis by washing them with a solution containing a substance capable ofdiffusing through the membrane of the red cell, such as glucose forexample.

FIG. 7 shows the lysis curves obtained after washing in solutionscontaining variable proportions of glucose and sodium chloride.

    ______________________________________                                        Prewashing solution (mos)                                                     ______________________________________                                        1 - 130 glycose 130 NaCl                                                      2 - 150 glucose 100 NaCl                                                      3 - 210 glucose  50 NaCl                                                      T -             300 NaCl                                                      ______________________________________                                    

The curves shown in FIG. 7 clearly demonstrate the advantage of swellingthe erythrocytes to improve lysis, although allowance must be made forthe fact that the volume of the erythrocytes also increases. For astarting haematocrit of 70%, the extracellular volume after resealingwill be greater if the mean cell volume has been artificially increased.Accordingly, these two effects are antagonistic so far as the finalencapsulation yield is concerned.

There is thus an optimum between the prewashing conditions and the lysisconditions, as illustrated in FIG. 8.

FIG. 8 shows the net and gross encapsulation yields for various bloodthroughputs and for the various prewashing solutions. It can be seenthat, for a given blood throughput, there is generally at least oneprewash which enables the yields to be increased.

The initial results given in Example 1 for the encapsulation of Desferalwere obtained using a chemical analysis technique. These results areoverestimated by comparison with those obtained with the radioactiveanalysis technique using the complex ⁵⁹ Fe-desferrioxamine used forobtaining the results shown in FIG. 6.

This Example shows that it is possible by adjusting the experimentalconditions to obtain results of from 40 to 50% for the gross yields.

EXAMPLE 8 Influence of the Quantity of Desferal added

In this Example, the quantity of Desferal encapsulated is measured forvarious concentrations of Desferal in the cell residue.

The test conditions are selected to give a gross yield of approximately40%.

FIG. 9 shows the results obtained and indicates the existence of alinear relation for a wide concentration range of Desferal.

It can be seen from FIG. 9 that the maximum dose of Desferal which canbe encapsulated is not reached. Using the optimal conditions shown inFIG. 6 or those corresponding to the use of two dialyzers as mentionedhereinafter (yield approximately 50%), it can be seen that it ispossible to encapsulate at least 800 mg to 1 g of Desferal per 100 ml ofresealed red blood cells (RBC).

EXAMPLE 9 Influence of Haematocrit

The following Table shows the effect of the haematocrit of the washedRBC on the encapsulation yield

    ______________________________________                                        Haematocrit %   Yield                                                         ______________________________________                                        50              35.3                                                          60              35.7                                                          70              40.4                                                          80              42                                                            ______________________________________                                    

EXAMPLE 10 Influence of the Resealing Temperature

The Table in Example 6 shows the effect of the resealing temperatureupon the resealing and encapsulation yield for IHP.

Complementary tests carried out with Desferal showed that, between 37°and 42° C., the effect of temperature on the final yield is fairlymoderate whereas, by contrast, the lysis curves of the resealederythrocytes are considerably modified.

FIG. 10 shows the effect of the resealing temperature upon osmotic lysisas a function of the isotonic strength of the medium.

The most stable forms are obtained in the region of 40° to 42° C.(displacement of the curves to the left). Beyond 45° C., the red cellsbecome unstable (displacement of the curves towards the right).

EXAMPLE 11

The composition of the resealing solution used in Example 1 plays animportant part, as shown in the following Table for two differenterythrocytes:

    ______________________________________                                        Composition of the solution                                                                     Gross yield %                                                                             Net yield %                                     ______________________________________                                        1 M NaCl          42.7-40     70.7-63.1                                       1.2 M NaCl        38.3-39.4   69.1-62.9                                       1.5 M Nacl        38.8-37.3   66.6-57.3                                       1 M NaCl + 10% PEG 4000                                                                         42.1-41.6   66.9-63.4                                       1.2 M NaCl + 10% PEG 4000                                                                       39.7-37.8   69.9-62.6                                       1.5 M NaCl + 10% PEG 4000                                                                       38.3-37.3   68.3-61.2                                       ______________________________________                                    

The dosages are made by isotopic method.

As mentioned above, it is well known that certain constituents areessential for keeping to the maximum life span of erythrocytes in vivo.This is the case with ATP, the Na/K equilibrium, Ca²⁺ and Mg²⁺ ions,glucose or other compounds, such as for example inosine or 2,3-DPG.

Accordingly, it may be necessary to modify the composition of resealingsolution or of the washing solutions in order to maintain the biologicalintegrity of the resealed erythrocytes intact.

EXAMPLE 12

This Example was carried out using an apparatus of the type illustratedin FIG. 3, i.e. an apparatus in which the active substance is introducedbetween two lysis phases of the red cells.

The following Tables show the average results obtained where thisapparatus is used for the encapsulation of Desferal.

The parameters are as follows:

S₁ =surface area of the dialyzer 1

S₂ =surface area of the dialyzer 28

blood flow rate=20, 40, 60 or 80 ml/minute

for different erythrocyte prewashing solutions;

(a) 0.15M NaCl solution (300 mos)

(b) NaCl solution, 130 mos+glucose, 130 mos.

    ______________________________________                                        Flow rate ml/min                                                                            Net yield %                                                                              Gross yield %                                        ______________________________________                                        (a) Prewashing with NaCl, 300 mos:                                            S.sub.1 = 0.28 m.sup.2 S.sub.2 = 0.28 m.sup.2                                 20            51.9       38.4                                                 40            38.8       31.6                                                 60            27.7       24.7                                                 80            19.3       17.7                                                 S.sub.1 = 0.28 m.sup.2 S.sub.2 = 0.41 m.sup.2                                 20            51.6       32.3                                                 40            39.6       28.7                                                 60            36.6       28.3                                                 80            38.1       27.5                                                 S.sub.1 = 0.41 m.sup.2 S.sub.2 = 0.28 m.sup.2                                 20            50.75      46.2                                                 40            50.57      39.5                                                 60            42.4       39.2                                                 80            34.4       32.6                                                 (b) Prewashing with NaCl, 130 mos + glucose 130 mos:                          S.sub.1 = 0.28 m.sup.2 S.sub.2 = 0.28 m.sup.2                                 20            51.8       46.8                                                 40            52         45.75                                                60            48.5       42.1                                                 80            51.5       45.7                                                 S.sub.1 = 0.28 m.sup.2 S.sub.2 = 0.41 m.sup.2                                 20            43.1       34.2                                                 40            50.4       46.2                                                 60            45.4       45.1                                                 80            46.6       39.7                                                 S.sub.1 = 0.41 m.sup.2 S.sub.2 = 0.28 m.sup.2                                 20            37.45      27.4                                                 40            38.7       37.7                                                 60            38         37.8                                                 80            38         37.15                                                ______________________________________                                    

The results thus obtained, compared with those shown in FIG. 8, indicatethat, under certain optimal test conditions, it is possible to improvethe encapsulation yield of a dialyzable substance and that these valuescorrespond to a final yield of from 45 to 50%, depending on the red cellused. Similarly, the net and gross yield values are closer due to thereduction in the loss of dialyzable substance. Optimal conditions may beestablished by trial and error for different values of S₁ and S₂.

EXAMPLE 13 Influence of the age of the red cell

The tests carried out showed that there is no significant variation inthe encapsulation yield when the erythrocytes used were less than 5days. Thereafter there is a progressive and slow reduction in resealingyield.

The following Examples show the principal test results obtained with thered cells containing IHP encapsulated by the process of Example 5.

EXAMPLE 14

FIG. 11 shows the relationship obtained between the P₅₀ -value of thesaturation curve and the concentration of IHP incorporated in human redcells. The correlation coefficient is 0.916.

The curves of FIG. 12 show the changes in the saturation of haemoglobinas a function of the oxygen pressure.

A study of the curves in FIG. 12 shows that, despite a reduction in thesaturation of haemoglobin for an arterial pressure of 100 mm Hg, thearteriovenous difference obtained at 30 and 40 mm is very significantlyincreased. These differences are very considerable for a displacement ofonly 10 to 20 min of the P₅₀ -values of the saturation curves.

EXAMPLE 15

Pig cells transformed under the same conditions as the human cells ofExample 14 were transfused to animals (piglets) byexsanguinotransfusion. Volumes of 300 to 500 ml reconstituted withhaematocrit from the haematocrit of the animal were used in each test.

The acid-base equilibrium and the oxygenation parameters before andafter transfusion are shown in the following Table:

    ______________________________________                                                   Before transfusion                                                                       After transfusion                                       ______________________________________                                        P.sub.50 mmHg                                                                              30.7 ± 1.0                                                                               41.2 ± 4.0***                                   pHa           7.36 ± 0.05                                                                            7.34 ± 0.04                                      pH-v          7.33 ± 0.05                                                                            7.28 ± 0.05                                      PaCO.sub.2 mmHg                                                                            39.0 ± 3.2                                                                              35.8 ± 5.4                                       PvCO.sub.2 mmHg                                                                            48.1 ± 6.5                                                                              45.3 ± 10.5                                      CO.sub.2 Ta mm/l                                                                           23.3 ± 3.5                                                                              19.0 ± 4.8                                       CO.sub.2 T-v mm/l                                                                          25.9 ± 3.6                                                                              21.3 ± 5.8                                       Hb g/100 ml  11.1 ± 2.0                                                                              11.2 ± 1.5                                       ______________________________________                                         ***P < 0.001                                                             

The transport of oxygen and the supply of oxygen to the tissues beforeand after transfusion are shown in the following Table:

    ______________________________________                                                  Before transfusion                                                                        After transfusion                                       ______________________________________                                        PaO.sub.2 mmHg                                                                            82.5 ± 10.3                                                                               97.5 ± 10.6*                                    PvO.sub.2 mmHg                                                                            36.7 ± 5.3 35.1 ± 3.7                                       CaO.sub.2 ml/100 ml                                                                       13.9 ± 2.5 14.1 ± 1.5                                       Cv O.sub.2 ml/100 ml                                                                      9.2 ± 2.9    5.6 ± 1.7***                                   DAV ml/100 ml                                                                             4.8 ± 1.1    7.6 ± 0.3***                                   Q l/mn/Kg   0.210 ± 0.029                                                                              0.144 ± 0.054***                               VO.sub.2 ml/mn/Kg                                                                         10.3 ± 1.6 10.6 ± 3.6                                       Part mmHg   91.5 ± 10.2                                                                               87.2 ± 22.8                                     ______________________________________                                         *P < 0.05                                                                     ***P < 0.001                                                             

These results show that, for a reduction in the affinity of haemoglobinfor oxygen, the entire animal responds by a reduction in cardiac outputand by an increase in its arteriovenous difference. The supply of oxygento the tissues is thus increased in relation to the transport of oxygen.FIG. 13 shows the trend followed by cardiac output as a function of theP₅₀ -value in 6 pigs exsanguinotransfused with pig cells in whichvariable quantities of IHP have been incorporated.

EXAMPLE 16 Characterization of the resealed erythrocytes (1) Electronmicroscope

FIG. 14, which is a photograph taken with an electron scan microscope,shows the comparative morphologies of the slightly echinocytic controlerythrocytes and of the lyzed and resealed erythrocytes which arecomparable in size. The echinocytic tendency is reduced. The cells areessentially stomatocytes of type I or II. 5% to 10% of the population issphero-echinocytic or vesicular.

(2) Haematological characteristics

FIG. 15 compares the erythrocyte volume distribution of washed andpacked RB C (curve a), as measured with a Coulter S, with that measuredafter resealing (curve b). A slight shift in the distribution towardsthe microcytic forms is observed.

The following Table shows the principal characteristics of theseerythrocytes.

    ______________________________________                                                (Man) (4)      Pig (2)                                                                                     Trans-                                           Control transformed                                                                              Control   formed                                   ______________________________________                                        Mean cell 94.3 ± 4                                                                             84 ± 3.1                                                                              59      52.2                                   volume                                                                        (10.sup.-6 m.sup.3)                                                           Mean cell 32.3 ± 1.9                                                                           30 ± 2.5                                                                              31      32.2                                   haemo-                                                                        globin conc.                                                                  (G/100 ml)                                                                    Cell distribution                                                                       15.4 ± 2.8                                                                           23.1 ± 7.2                                                                            21.3    29.2                                   index                                                                         ______________________________________                                    

It can be seen from these results that the erythrocytes lyzed andresealed by the process according to the invention are slightlymicrocytic and normochromic with an enlarged volume distribution towardsthe low values.

(3) Immunological characteristics

The lyzed and resealed erythrocytes were examined for qualitative andquantitative changes in the antigens of blood groups. The conventionalmethods of immunohaemotology were used, including Coombs' tests andtreatment with proteolytic enzymes.

The aggregates obtained are slightly reduced in size, but are completeand perfectly visible. Accordingly, erythrocytes such as these, aftertransformation, may be compatibilized with regard to a potentialreceiver.

No significant quantitative or qualitative change was observed forerythrocytes having incorporated Desferal in the following antigensystems: ABO-Rh-CcDEe-Kk kpa kpb-MNSs-P₁ -le^(a) -le^(b) -Fy^(a) -Fy^(b)-JK^(a) -JK^(b) -Lu^(a).

The same applies to the following antigens: Ku-Gerbich-Fy³ -JK³, and tothe P antigens--the Cartwright and Vel antigens.

No anomaly in the polyagglutinability antigens is observed in AB serum.

These results are a clear indication of the nonmodification of thepublic, private or polyagglutinability antigens for the transformederythrocytes. This result is essential to the use in vivo of theseerythrocytes for transporting substances of biological interest,particularly immunomodulators which are capable of acting on the immuneresponse of the receiver.

EXAMPLE 17 Encapsulation of MDP and its derivatives

Among the numerous synthetic or semisynthetic immunomodulators, thegroup of muramyl dipeptides appears particularly promising.

It has been found that some of these substances can be encapsulated bythe process according to the invention.

A study has been made of their stability as a function of time inerythrocytes stored at 4° C.

100 mg of murabutide (butyl MDP) (GIRPI) in 10 ml of physiological serumare mixed with 200 ml of washed cell residue containing 70% ofhaematocrit. After lysis and resealing by the process of Example 2, 23mg (23%) are found in 96 ml of washed and resealed RBC having 64% ofhaematocrit.

Over a period of 8 days, no significant change in the concentration ofthis substance in the erythrocytes stored at 4° C. is observed by HPLCanalysis which signifies that this type of peptide compound is notdegraded in the erythrocytes during storage, in contrast to insulin, inthe absence of protease inhibitors.

Over the same period, no significant concentration of this MDPderivative is observed outside the red cells which indicates that thereis no significant transmembran loss of this compound. The encapsulationyield is relatively low (23%) which may be attributed to the hydrophobicnature of the compound and to it interaction with the erythrocytemembrane.

A normal therapeutic dose of 5 mg, as currently estimated, would onlynecessitate about 20 ml of tranformed blood, which indicates the extremeeffectiveness of this mode of transport. An operation such as this makesit possible very easily to obtain 5 useful doses spread over a givenperiod for the same patient. In addition, this example does not in anyway limit the encapsulable dose (approximately 1 g/100 ml for Desferal).

EXAMPLE 18 Encapsulation of α interferon

Following exactly the same procedure as in Example 17, a solution ofpurified α-interferon containing 2.1·10⁶ units in 1 ml is mixed with 220ml of cell residue. In view of the very low protein concentration, thedialysis circuit is incubated for 30 minutes beforehand with a 1% humanalbumin solution to limit the losses by nonspecific absorption in thedialysis circuit. After lysis and resealing, 840,000 units of interferon(38% yield) are found in the 125 ml of resealed and washed cell residue.It is pointed out that this yield is similar to that obtained foralbumin.

Within the limits of the biological titration conducted, there is noclear reduction in the strength of the interferon over a period of 8days at 4° C. after its encapsulation in the erythrocytes which isindicative of the excellent preservation of this compound in vivo afterits encapsulation.

Encapsulation yield of the same magnitude are observed with γ interferonand interleukine II.

We claim:
 1. An apparatus for the encapsulation in human or animalerythrocytes of at least one substance having a biological activity,comprisinga first dialysis element comprising at least one first primarycompartment and one first secondary compartment which are separated by adialysis membrane, a means for the continuous supply of an erythrocytesuspension into the primary compartment of said dialysis element, ameans for supplying a hypotonic aqueous solution into the secondarycompartment of the dialysis element, a means for supplying the substancehaving a biological activity into the primary compartment of thedialysis element, and a means for transferring an effluent solution fromthe primary compartment into a resealing assembly, the resealingassembly comprising at least one means for increasing the osmosticand/or oncotic pressure of the effluent solution.
 2. An apparatusaccording to claim 1, wherein the resealing assembly comprises a seconddialysis element comprising at least one primary compartment and onesecondary compartment which are separated by a dialysis membrane, theeffluent solution being supplied into the primary compartment of saidsecond dialysis element by the transfer means and the means forincreasing the osmotic and/or oncotic pressure comprises a solutionwhich is hypertonic with respect to the effluent solution, saidhypertonic solution being supplied into the secondary compartment ofsaid second dialysis element.
 3. An apparatus according to claim 1,wherein the resealing assembly comprises an enclosure containing a meansfor supplying a solution which is hypertonic with respect to theeffluent solution.
 4. An apparatus according to claim 1, which furthercomprises a heating means disposed between the first dialysis elementand the resealing assembly operable to heat the fluid from the firstdialysis element.
 5. An apparatus according to claim 1, wherein theresealing assembly is provided with a heating means to heat the fluidwithin the resealing assembly.
 6. An apparatus according to claim 1,which further comprises a means for controlling the osmotic pressure ofthe erythrocyte suspension.
 7. An apparatus according to claim 1, whichfurther comprises pump means for controlling the pressure of the fluidscirculating in the apparatus.
 8. An apparatus as claimed in claim 1,further comprising an additional dialysis element comprising at least asecond primary compartment and a second secondary compartment separatedby a dialysis wall, said second primary compartment in downstreamcommunication with said first primary compartment, and said secondsecondary compartment in upstream communication with said firstsecondary compartment.
 9. An apparatus according to claim 1 furthercomprising a receiver, said receiver being disposed after the firstdialysis element.
 10. An apparatus according to claim 8, whereinperistaltic pump means moves said suspension through said primarycompartments.